Although the structure of erythrocyte spectrin, actin, and proteins 3 and 4.1 are now known in great detail, this is not so for other erythrocyte membrane proteins including ankyrin and protein 4.9a. No published sequence information exists for either of these proteins and little is known about the binding interactions of 4.9a. We plan to undertake detailed studies of both of them, so that by the end of this grant period (or the start of the next one) it will be possible to begin more detailed investigations of these proteins in nonerythroid cells. In addition we will establish whether some patients with hereditary spherocytosis have a defect in ankyrin, as preliminary studies suggest, and define this defect(s). Specifically we will: 1. Clone and sequence full length cDNAs for human erythrocyte ankyrin and protein 4.9a. Analyze the structure of both proteins, determine the chromosomal location of the corresponding genes, and identify polymorphic restriction sites (RFLPs). 2. Test the hypothesis that some families with dominant hereditary spherocytosis (HS) have a deficiency or functional defect in ankyrin. Screen patients from dominant HS families for: ankyrin deficiency, abnormal ankyrin-spectrin interactions, and linkage between HS and ankyrin (using RFLP patterns). Further investigate """"""""positives"""""""" by: (a) mapping ankyrin domains; (b) measuring ankyrin functions, including spectrin, protein 3, protein 4.2, tubulin, and vimentin interactions, and the binding of normal ankyrin by HS proteins 3 and 4.2; (c) testing ankyrin phosphorylation by cAMP- dependent and casein kinases; and (d) analyzing ankyrin gene structure by Southern blotting. 3. Investigate (a) the domain structure of normal protein 4.9a, and its functions, including: (b) its self-association; (c) its membrane binding site; (d) its interaction with proteins that form the skeletal junction (actin, spectrin, 4.2, tropomyosin, and adducing); and (e) the effect of phosphorylation on these associations. 4. Determine the expression of ankyrin and protein 4.9a in various tissues and cells by Northern and Western blotting, and localize these proteins by immunofluorescence. 5. Clone and sequence the """"""""mini""""""""(72kD)-ankyrin present in B and T lymphocytes and platelets or, perhaps, another nonerythroid ankyrin defined by the expression and immunofluorescence studies described in the previous paragraph.
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